lab-on-a-chip part 2 – detection methodslab-on-a-chip part 2 – detection methods ......

Introduction to BioMEMS & Medical Microdevices

Lab-on-a-Chip Part 2 – Detection Methods Prof. Steven S. Saliterman, http://saliterman.umn.edu/

Steven S. Saliterman

Topics

Electrochemical Detection Capillary electrophoresis. Electrochemical

impedance spectroscopy (EIS).

Optical Detection Conventional Off-Chip or

“Free-Space” Absorbance Detection Fluorescence Detection Chemiluminescence Surface Plasmon

Resonance Surface Enhanced

Raman Spectroscopy

On-Chip Methods Frequency Specific LEDs Absorbance

Spectroscopy Fluorescence Chemiluminescence Optical Waveguides Surface Plasmon

Resonance Interferometry Holography

Steven S. Saliterman Images courtesy of Micronit

Glass and Polymer Based Devices

http://www.micronit.com/pressure_chip_holder_for_microfluidic_glass_chips.cfm


Positive “Downscaling” Effects

Reduced sample and reagent volumes. Ten fold reduction in length scale. Laminar flow (low Reynolds numbers). Improved mass flow by diffusion. Harvesting electrokinetic effects The surface-to-volume ratio (SVR) may increase by a

factor of more than ten thousand during downscaling and solute/wall interactions become dominant.


Consider Capillary Electrophoresis

Guber AE at al., Microfluidic lab-on-a-chip systems based on polymers – fabrication and application.” Chemical Engineering Journal 101(1:3), pp 447-453 (2004). Image courtesy of Micronit.

a) Sample introduction and electrophoretic separation are accomplished in each of two crossing channels.

b) Sample is driven through the short sample channel across the separation channel by application of a potential, and

c) The introduced sample “plug” (nanoliter volume) is then electrophoretically separated by application of another potential.


Capillary Electrophoresis…

Gencoglu, A and Adrienne R. Minerick . Electrochemical detection techniques in micro- and nanofluidic devices. Microfluid Nanofluid (2014) 17:781–807

a) High voltage along the length of the capillary separates the analytes and drives them toward the detection electrodes.

b) A constant potential is applied with a potentiostat to the detection electrodes. c) As each species passes, a change in current or conductivity between the detection electrodes is

detected.

a)

b)

c)


Electrochemical Impedance Spectroscopy

a) Three-electrode electrochemical cell. (WE = Working Electrode, CE = Counter Electrode, RE = Reference Electrode & V = Voltage).

b) Equivalent circuit diagram. (R = Resistance, DL = Double Layer, S = Series Resistance. Rohm = Resistance of the Bulk Solution.

a) Gencoglu, A and Adrienne R. Minerick . Electrochemical detection techniques in micro- and nanofluidic devices. Microfluid Nanofluid (2014) 17:781–807 b) Fan L, Zhao G, Shi H, Liu M, Li Z (2013) A highly selective electrochemical impedance spectroscopy-based aptasensor for sensitive detection of acetamiprid. Biosens Bioelectron 43:12–18. doi:10.1016/j.bios.2012.11.033


Recall Aptamers…

Image courtesy of Archemix

Aptamers are artificial nucleic acid ligands that can be generated against amino acids, drugs, proteins and other molecules.

Function similar to antibodies. Applications:

Therapeutics, Target validation, Drug screening, Affinity separation, Diagnostics and biosensors.


Example of EIS Implementation…

a) Gencoglu, A and Adrienne R. Minerick . Electrochemical detection techniques in micro- and nanofluidic devices. Microfluid Nanofluid (2014) 17:781–807 b) Fan L, Zhao G, Shi H, Liu M, Li Z (2013) A highly selective electrochemical impedance spectroscopy-based aptasensor for sensitive detection of acetamiprid. Biosens Bioelectron 43:12–18. doi:10.1016/j.bios.2012.11.033

Nyquist plots obtained with EIS.


Optical Detection: Off-Chip

Absorbance Detection Fluorescence Detection Chemiluminescence Surface Plasmon Resonance Localized Surface Plasmon Resonance (LSPR)

Spectroscopy (electromagnetic-field enhancement that leads to surface enhanced Raman Scattering and other surface-enhanced processes)

Gai, Hongwei, Yongjun Li, and Edward S. Yeung. 2011. Optical Detection Systems on Microfluidic Chips. Microfluidics: Technologies and Applications 304, 171-201.


Absorbance Detection

UV/Vis light is used for absorption spectroscopy. Absorption spectra are related to the structure and concentration

of the analyte and are based on the capability of samples to attenuate the incident electromagnetic radiation at various wavelengths.


Introducing Fluorophores…

A fluorophore is a fluorescent chemical compound that may re-emit light upon light excitation.

Fluorophores typically contain several combined aromatic groups, or plane or cyclic molecules with several π bonds.

Generally covalently bonded to a macromolecule, serving as a marker (or dye, or tag, or reporter) for affine or bioactive reagents (antibodies, peptides, nucleic acids).

Fluorophores are notably used to stain tissues, cells, or materials in a variety of analytical methods.

Wikipedia.org

Nat. Inst. of General Medical Sciences, NIH

Excitation

Emission


Fluorophore Characteristics

Maximum excitation and emission wavelength (nm): Corresponds to the peak in the excitation and emission

Extinction Coefficient (Mol−1cm−1) : Links the quantity of absorbed light, at a given wavelength, to the

concentration of fluorophore in solution. Quantum yield (emitted/absorbed photons):

Efficiency of the energy transferred from incident light to emitted fluorescence .

Lifetime (in picoseconds): Duration of the excited state of a fluorophore before returning to its ground

state Stokes shift:

Difference between the max excitation and max emission wavelengths.

Wikipedia.org


Introducing Molecular Beacons…

Single-stranded oligonucleotide hybridization probes that form a stem-and-loop structure:


Target DNA

Fluorophore

Quencher

Molecular Beacon

Public Health Research Institute

Molecular Beacons…


Confocal Scanning Laser Microscopy…

Human Medulla

Rabbit Muscle Fiber

Sunflower Pollen Grain

Image courtesy of Olympus


Chemiluminescence

Chemiluminescence (CL) is the generation of light (visible, ultraviolet and infrared) by the release of energy from a chemical reaction.

Advantages: No excitation source (as does fluorescence and phosphorescence), Only a single light detector such as a photomultiplier tube, No monochromator and often not even a filter.

Detection limits can be 10 to 100 times lower than other luminescence techniques.

Peroxyoxalates are esters formed by the reaction of hydrogen peroxide and oxalate esters.

Chasteen, TG


Peroxyoxalate “light-stick” reaction and emission from the reaction of luminol with hydrogen peroxide in basic aqueous solution catalyzed by cobalt II:

Image courtesy of Simon W. Lewis, Deakin University

Peroxyoxalate Chemiluminescence


Introducing Surface Plasmons…

Coherent delocalized electron oscillations that exist at the interface between any two materials.

Surface plasmon polaritons are surface electromagnetic waves that propagate in a direction parallel to the metal/dielectric (or metal/vacuum) interface.

Since the wave is on the boundary of the metal and the external medium (air or water for example), these oscillations are very sensitive to any change of this boundary, such as the adsorption of molecules to the metal surface

Wikipedia


Surface Plasmon Resonance

Wikipedia

Resonant oscillation of conduction electrons at the interface between a negative and positive permittivity material stimulated by incident light. Detection is possible because adsorbing molecules cause changes in the local index of refraction, changing the resonance conditions of the surface plasmon waves. Basis for measuring adsorption of material onto planar metal (typically gold and silver) surfaces or onto the surface of metal nanoparticles. It is the fundamental principle behind many color-based biosensor applications and different lab-on-a-chip sensors. Reflection/Absorption/Reflection

Flow Cell

Single wavelength


“Localized” Surface Plasmon Resonance

a) A surface plasmon polariton (or propagating plasmon)

b) A localized surface plasmon. Light interacts with particles much smaller than the incident wavelength. This leads to a plasmon that oscillates locally around the nanoparticle with a frequency known as the LSPR. Similar to the SPR, the LSPR is sensitive to changes in the local dielectric environment.

Willets, Katherine A. And RP Van Duyne . Localized surface plasmon rsonance spectroscopy and sensing Annu. Rev. Phys. Chem. 2007. 58:267–97

Au Nanoparticles


Localized SPR Explained...

Localized surface plasmon resonance (LSPR) spectroscopy of metallic nanoparticles is a powerful technique for chemical and biological sensing experiments

Materials that possess a negative real and small positive imaginary dielectric constant are capable of supporting a surface plasmon resonance (SPR).

Plasmonics is the study of these particular light-matter interactions

Willets, Katherine A. And RP Van Duyne . Localized surface plasmon rsonance spectroscopy and sensing Annu. Rev. Phys. Chem. 2007. 58:267–97


Example: Cancer Marker Detection – Acimovic et. al.

Gold Nanorod Array

Acimovic, SS. LSPR Chip for Parallel, Rapid, and Sensitive Detection of Cancer Markers in Serum. Nano Lett. 2014, 14, 2636−2641


Sensing Approach

Schematic of the sensing approaches used for the detection of the analyte of interest where : a) A biotin−avidin is used to link the receptor. b) Carbodiimide chemistry is used.

Gold Nanorods



Detection of AFP and PSA Markers

Parallel biosensor chip (LSPR response) for the detection of AFP and PSA cancer markers in 50% human serum.



Optical Detection: On-Chip

Frequency Specific LEDs Absorbance Spectroscopy Fluorescence Chemiluminescence Optical Waveguides Surface Plasmon Resonance Interferometry Holography


Introducing LEDs…

Image Courtesy of Zeiss


Absorbance Spectroscopy On-Chip

Increasing optical path length by making the light pass axially between parallel 45 degree mirrors.

Noda T, Takao H, Yoshioka K, Oku N, Ashiki M, Sawada K, Matsumoto K, Ishida M(2006) Sens Actuators B 119:245–250



Fluorescence

Shen, L.; Ratterman, M.; Klotzkin, D.; Papautsky, I. A CMOS optical detection system for point-of-use luminescent oxygen sensing. Sens. Actuators B Chem. 2011, 155, 430–435.

Conceptual design of a fluorescence based detection device showing a light source (LED), photodetector (CMOS), polarizers and O2 sensitive PtOEP film arranged in a portable O2 sensing system.


Chemiluminescence

Integrated opto-microfluidic sensor with a hydrogenated amorphous silicon (a-Si:H) photodetector prepared onto a glass substrate covered by a transparent conductive oxide (TCO) film.

Caputo, D.; de Cesare, G.; Dolci, L.S.; Mirasoli, M.; Nascetti, A.; Roda, A.; Scipinotti, R. Microfluidic chip with integrated a-Si:H photodiodes for chemiluminescence-based bioassays. IEEE Sens. J. 2013, 13, 2595–2602.


Waveguides for LOC based on Refractive Index

1. Solid-state waveguides. Solid fibers (silica, glass or polymer) enter the chip and intersect the fluidic

channel, exciting analytes and collecting the response. 2. Liquid-core waveguides (LCWs).

The microchannel not only transports the sample, but also transmits the light.

If liquid (particularly water in a microfluidic chip, with refractive index of 1.33) is used as the core of waveguide, the refractive indices of the cladding should be smaller.

3. Liquid-core/liquid-cladding (L2) waveguides Two or more different laminar liquids of different refractive index flowing

inside a fluidic channel. The index of the cladding liquid is smaller than that of the core liquid so that

the light is guided in the channel by the total internal reflection.


Steven S. Saliterman Ligler FS (2009) Anal Chem 81:519–526

Liquid-Core/ Cladding (L2) Waveguide


Three parallel waveguides formed with liquid core and cladding in laminar flow systems. The direction of the light propagation can be altered by different flow rates of the adjacent fluids.


Surface Plasmon Resonance On-Chip

Coupling schemes of incident light to surface plasmons.

Gencoglu, Aytug, and Adrienne R. Minerick. 2014. Electrochemical detection techniques in micro- and nanofluidic devices. Microfluidics and Nanofluidics 17, no. 5:781-807.

Traditional Prism Coupling

Fiber Optic SPR Sensor

Waveguide Coupling Grating Coupling


Recall Interferometry…

Physics Lab Online and Lenox Laser

Young’s Double Slit Interferometer

Diffraction


Mach-Zehnder Interferometer (MZI)

Gonzalez-Guerrero, A. B., et al. 2011. Advanced photonic biosensors for point-of-care diagnostics. Eurosensors Xxv 25,


Digital In-Line Holographic Microscope

Wu, J at al. Optical imaging techniques in microfluidics and their application. Lab Chip, 2012, 12, 3566–3575


Summary

Electrochemical Detection Capillary electrophoresis. Electrochemical

impedance spectroscopy (EIS).

Optical Detection Conventional Off-Chip or

“Free-Space” Absorbance Detection Fluorescence Detection Chemiluminescence Surface Plasmon

Resonance Surface Enhanced

Raman Spectroscopy

On-Chip Methods Frequency Specific LEDs Absorbance

Spectroscopy Fluorescence Chemiluminescence Optical Waveguides Surface Plasmon

Resonance Interferometry Holography

Appendix Tables of optical

detection methods


Table of Chemiluminescence Detection

Pires, NM, et al. Recent Developments in Optical Detection Technologies in Lab-on-a-Chip Devices for Biosensing Applications. Sensors 2014, 14, 15458-15479


52. Xiang, A.; Wei, F.; Lei, X.; Liu, Y.; Liu, Y.; Guo, Y. A simple and rapid capillary chemiluminescence immunoassay for quantitatively detecting human serum HBsAg. Eur. J. Clin. Microbiol. Infect. Dis. 2013, 32, 1557–1564.

53. Hao, M.; Ma, Z. An ultrasensitive chemiluminescence biosensor for carcinoembryonic antigen based on autocatalytic enlargement of immunogold nanoprobes. Sensors 2012, 12, 17320–17329.

54. Yang, M.; Sun, S.; Kostov, Y.; Rasooly, A. An automated point-of-care system for immunodetection of staphylococcal enterotoxin B. Anal. Biochem. 2011, 416, 74–81.

55. Caputo, D.; de Cesare, G.; Dolci, L.S.; Mirasoli, M.; Nascetti, A.; Roda, A.; Scipinotti, R. Microfluidic chip with integrated a-Si:H photodiodes for chemiluminescence-based bioassays. IEEE Sens. J. 2013, 13, 2595–2602.

56. Lin, C.C.; Ko, F.H.; Chen, C.C.; Yang, Y.S.; Chang, F.C.; Wu, C.S. Miniaturized metal semiconductor metal photocurrent system for biomolecular sensing via chemiluminescence. Electrophoresis 2009, 30, 3189–3197.

57. Wojciechowski, J.R.; Shriver-Lake, L.C.; Yamaguchi, M.Y.; Füreder, E.; Pieler, R.; Schamesberger, M.; Winder, C.; Prall, H.J.; Sonnleitner, M.; Ligler, F.S. Organic photodiodes for biosensor miniaturization. Anal. Chem. 2009, 81, 3455–3461.


Table of Fluorescence Detection

Pires, NM, et al. Recent Developments in Optical Detection Technologies in Lab-on-a-Chip Devices for Biosensing Applications. Sensors 2014, 14, 15458-15479


41. Lee, L.M.; Cui, X.; Yang, C. The application of on-chip optofluidic microscopy for imaging Giardia lamblia trophozoites and cysts. Biomed. Microdevices 2009, 11, 951–958.

49. Ramalingam, N.; Rui, Z.; Liu, H.B.; Dai, C.C.; Kaushik, R.; Ratnaharika, B.M; Gong, H.Q. Real-time PCR-based microfluidic array chip for simultaneous detection of multiple waterborne pathogens. Sens. Actuators B Chem. 2010, 145, 543–552.

50. Yildirim, N.; Long, F.; Gao, C.; He, M.; Shi, H.C.; Gu, A.Z. Aptamer-based optical biosensor for rapid and sensitive detection of 17β-estradiol in water samples. Environ. Sci. Technol. 2012, 46, 3288–3294.

51. Ishimatsu, R.; Naruse, A.; Liu, R.; Nakano, K.; Yahiro, M.; Adachi, C.; Imato, T. An organic thin film photodiode as a portable photodetector for the detection of alkylphenol polyethoxylates by a flow fluorescence-immunoassay on magnetic microbeads in a microchannel. Talanta 2013, 117, 139–145.

lab-on-a-chip part 2 – detection methodslab-on-a-chip part 2 – detection methods ......

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